In mammalian cells, the proteins Mre11, RadSO and nibrin form the MRN complex which acts both in sensing the presence of DNA double-strand breaks (DSB) and in facilitating signaling by ATM to downstream effectors that activate cellular cycle checkpoint and repair mechanisms. Mutations in any of the three components of this complex result in syndromes characterized by hypersensitivity to ionizing radiation (IR), an increased incidence of cancer, immunodeficiency, and an overlapping set of growth and developmental defects. Mre11 and RadSO are essential genes, and the high degree of evolutionary conservation in the proteins they encode allow inferences regarding their function and structure based on data from homologues in other more experimentally tractable organisms. Nibrin, however, is considerably less well conserved and its function is, accordingly, less well understood. We originally identified the NBS1 gene that encodes nibrin through genetic linkage studies in NBS families. Subsequently, we have utilized the approach of site-specific mutagenesis of human nibrin and transfection into NBS patient cell lines to identify functional domains and evaluate their roles in the cellular response to IR exposure. This approach has allowed us to identify and characterize several distinct domains of nibrin that function in the mammalian DNA damage response, as well as novel functions and new interacting partners that await characterization. However, this approach is limited by the hypomorphic nature of all known human NBS mutations that results in the synthesis of a truncated protein providing partial nibrin function in NBS cell lines. This same effect also limits the informativeness of studies of the role of nibrin in development since homozygosity for knockout alleles in mice results in embryonic lethality. We have now developed cell line and whole animal models based on conditional gene inactivation that will allow us to study the role of nibrin in cellular DNA damage responses and in mammalian development in a null background. We propose to use these systems to explore the role of nibrin in sensing DNA damage and activating DNA damage responses, in protecting telomeres during the course of damage responses, in downregulating damage response pathways after completion of repair and in the development of the immune system. [unreadable] [unreadable] [unreadable]